The catastrophic complications due to deficiency in plasma metalloprotease (ADAMTS13) highlight the biological importance of this protease in regulation of hemostasis and prevention of thrombosis in microvascular circulation that occurs in the thrombotic thrombocytopenic purpura (TTP) syndrome. Identification of the gene encoding ADAMTS13 metalloprotease and determination of the structure elements in the encoded protein have opened a new era in understanding of the roles of a zinc metalloprotease in regulation of hemostasis and thrombosis, and provided the essential tools to address the fundamental (patho)biological questions concerning ADAMTS13 including its biosynthesis, activation, substrate recognition, intracellular sorting and autoantibody interaction. The goals of the proposed research are: Specific Aim 1. To determine ADAMTS13 substrate recognition by 1) analyzing the proteolytic activity of wild type ADAMTS13 and various mutants of ADAMTS13 toward von Willebrand factor and its analog, VWF73; 2) determining the binding kinetics between VWF or VWF73 and wild type or mutant ADAMTS13 by radioligand binding and surface plasmon resonance assays. Specific Aim 2. To determine intracellular sorting of ADAMTS13 by 1) determining the polarity of full-length and mutant ADAMTS13 secretion in MDCK cells; 2) determining the sorting signal and mechanisms by which ADAMTS13 is sorted; 3) determining the effect of point mutations or truncations of ADAMTS13 gene found in patients with congenital TTP on the polarity of ADAMTS13 secretion. Specific Aim 3. To determine the domains (or sites) of ADATMTS13 to which anti-ADAMTS13 autoantibodies bind and the kinetic parameters (affinity and specificity) of interaction between ADAMTS13 metalloprotease and anti-ADAMTS13 autoantibodies, and to correlate the clinical course and outcome to the distinct type of anti-ADAMTS autoantibodies identified in patients with TTP.